Formation and uses of europium

ABSTRACT

An MRI contrast composition includes a liposome and a europium metal complex disposed within the liposome. The europium metal complex includes a europium metal ion and a multi-dentate ligand selected from the group consisting of cryptands and thiacryptands and one or more counter-ions that balances a charge of the europium metal ion and the multi-dentate ligand, the europium metal ion being switchable between a 2+ and 3+ oxidation state. The contrast composition advantageously provides an oxidation-responsive dual-mode contrast agent because it would enhance either T 1 -weighted images or CEST images depending on the oxidation state of Eu.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. provisional Application No.61/776,354 filed Mar. 11, 2013, the disclosure of which is incorporatedin its entirety by reference herein.

TECHNICAL FIELD

In at least one aspect, the present invention is related to magneticresonance imaging contrast agents.

BACKGROUND

The power of magnetic resonance imaging (MRI) resides in the ability toascertain anatomical information at high resolution (<1 mm³). Molecularinformation can also be obtained with MRI using responsive paramagneticcomplexes (contrast agents) that alter water proton signal intensitiesin response to chemical events. Some contrast agents respond to changesin pH, temperature, or metal ion concentration; enzyme activity; thepresence of free radicals, antioxidants, phosphate diesters, or singletoxygen; or changes in the partial pressure of oxygen. Of particularinterest are targets that cause changes in redox behavior because theyare associated with cancer, inflammation, and cardiovascular diseases.Therefore, responsive contrast agents that target redox changes have thepotential to greatly improve the diagnostic capabilities of MRI.However, a critical limitation of responsive contrast agents thathinders their use in vivo is that determination of molecular informationrequires knowledge of the concentration of the contrast agents, which isexceedingly difficult to measure in vivo. Some systems have achievedconcentration independence in contrast-enhanced MRI through ratiometrictechniques (longitudinal vs transverse relaxation rates), ratiometricchemical exchange saturation transfer (CEST) techniques, or the use oforthogonal detection modes with a multimodal agent. However, no reportedsystems respond to general oxidizing events based on tunable redoxpotentials. An ideal metal for tunable multimodal redox response is Eubecause the Eu²⁺ and Eu³⁺ oxidation states differentially enhanceT₁-weighted and CEST images in MRI. Furthermore, Eu²⁺ has a tunableoxidation potential and outperforms clinically approved T₁-shorteningcontrast agents at ultra-high magnetic field strengths.

Accordingly, there is a need for improved contrast agents for magneticresonance imaging.

SUMMARY

In at least one embodiment, the present invention provides an MRIcontrast composition. The composition includes a liposome and a europiummetal complex disposed within the liposome. The europium metal complexincludes a europium metal ion and a multi-dentate ligand selected fromthe group consisting of cryptands and thiacryptands and if necessarycounter-ions to maintain charge neutrality (i.e., balances a charge ofthe europium metal ion and the multi-dentate ligand), the europium metalion being switchable between a 2+ and 3+ oxidation state. The presentembodiment advantageously provides an oxidation-responsive dual-modecontrast agent because it would enhance either T₁-weighted images orCEST images depending on the oxidation state of Eu.

In another embodiment, a MRI contrast compound is provided. The compoundis described by formula (II):

wherein:n, o are each independently 1 or 2;L₁ is a multi-dentate ligand moiety that bonds to Eu;L₂ is a multi-dentate ligand moiety that bonds to Gd;Xp are counter ions necessary to maintain charge neutrality; andSp is a spacer moiety that provides separation between Eu and Gd.Advantageously, compound II is useful as an MRI contrast agent whetheror not it is encapsulated in a liposome.

In still another embodiment, a europium metal complex having formula(VIII) is provided:

wherein:c is the charge of the combination of the europium metal atom and themulti-dentate ligand;Xp represents a number of counter-ions necessary for charge neutrality;Y¹, Y², Y², Y⁴, Y⁵, and Y⁶ are each independently O or S;R₁, R₂, R₃ are each independently H, C₁₋₁₂ alkyl, C₁₋₁₂ alkynyl, C₁₋₁₂alkenyl, C₁₋₁₂ fluoroalkyl, Cl, F, Br, nitro, cyano, or C₆₋₁₄ aryl,C₅₋₁₄ hetereoaryl, or 5 and 6 membered rings formed by combining R₁ onadjacent carbon atoms or R₂ and R₃ on adjacent carbon atoms, ═O bycombining R₁, R₂, or R₃ on the same carbon atom, ═S by combining R₁, R₂,or R₃ on the same carbon atom, or ═NR by combining R₁, R₂, or R₃ on thesame carbon atom;R is H or C₁₋₁₂ alkyl; andR₄, R₅, R₆, R₇ are each independently hydrogen, cyano, nitro, Cl, F, Br,I, CH₃, NH₂, C₆₋₁₅ aryl, carboxylated C₆₋₁₈ aryl, C₅₋₁₅ heteroaryl,carboxylated C₅₋₁₈ heteroaryl, C₁₋₁₂ alkyl, phenyl, carboxylated phenyl,carboxylated C₂₋₁₂ alkyl, —CO₂H, —CH₂CO₂H,

R₈, R₉, R₁₀, R₁₁, R₁₂ are each independently H or CO₂H. Advantageously,compound VIII is useful as an MRI contrast agent whether or not it isencapsulated in a liposome.

In another embodiment, a method of magnetic resonance imaging of anorgan or organ structure in a subject is provided. The method includes astep of administering a liposome composition to the subject. Theliposome composition includes a liposome and a europium metal complexdisposed within the liposome. The europium metal complex includes aeuropium metal ion and a first multi-dentate ligand selected from thegroup consisting of cryptands and thiacryptands and one or morecounter-ions that balance a charge of the europium metal ion. Images ofan organ in the subject are taken by magnetic resonance imagining.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 provides a schematic representation of the oxidation ofliposome-encapsulated Eu(2.2.2)2+ (T1 enhancement, CEST present) to forma liposome filled with Eu³⁺ (T1 silent, increased CEST enhancement). Onthe far right is a depiction of the liposomal phospholipid bilayer withovals as cholesterol molecules. For clarity, only one complex is shownin each liposome and coordinated water molecules are not drawn;

FIG. 2 provides oxidations potential for various europium/cryptandcomplexes;

FIG. 3 provides specific examples of cryptands and thiacryptands thatare useful as multi-dentate ligands in forming europium complexes;

FIG. 4 provides synthetic pathways for forming cryptands that are usefulas multi-dentate ligands in forming europium complexes;

FIG. 5 provides specific examples of cryptands and thiacryptands thatare useful as multi-dentate ligands in forming europium complexes;

FIG. 6 provides Lorentzian-fitted CEST spectra (7 T, ambienttemperature) of liposomes before (dashed line) and after (solid line)air exposure. Bulk water was referenced to 0 ppm and signal intensitieswere calculated from in vitro images after a 2 s presaturation with a 17μT radiofrequency pulse from 5 to −5 ppm in 0.2 ppm increments.

FIG. 7 provides a MTR_(asym) vs frequency offset of presaturation forliposomes before (dashed line) and after (solid line) air exposure.MTR_(asym) was calculated using fitted Lorentzian functions.

FIG. 8 provides MR phantom images (5 mm tube diameter, 7 T, and ambienttemperature) of water, non-oxidized liposomes containing Eu²⁺, andoxidized liposomes containing Eu³⁺. The top row contains T₁-weightedimages, and the bottom row contains CEST maps generated by subtractingpresaturation at 1.2 ppm from presaturation at −1.2 ppm and dividing thedifference by the presaturation at −1.2 ppm; and

FIG. 9 provides a plot of T1 vs time of the EuII-containing complex ofligand 4 (3 mM) in the absence (□) and presence (∘) of glutathione (325mM).

DETAILED DESCRIPTION

Reference will now be made in detail to presently preferredcompositions, embodiments and methods of the present invention whichconstitute the best modes of practicing the invention presently known tothe inventors. The Figures are not necessarily to scale. However, it isto be understood that the disclosed embodiments are merely exemplary ofthe invention that may be embodied in various and alternative forms.Therefore, specific details disclosed herein are not to be interpretedas limiting, but merely as a representative basis for any aspect of theinvention and/or as a representative basis for teaching one skilled inthe art to variously employ the present invention.

Except in the examples, or where otherwise expressly indicated, allnumerical quantities in this description indicating amounts of materialor conditions of reaction and/or use are to be understood as modified bythe word “about” in describing the broadest scope of the invention.Practice within the numerical limits stated is generally preferred.Also, unless expressly stated to the contrary: percent, “parts of,” andratio values are by weight; the description of a group or class ofmaterials as suitable or preferred for a given purpose in connectionwith the invention implies that mixtures of any two or more of themembers of the group or class are equally suitable or preferred;description of constituents in chemical terms refers to the constituentsat the time of addition to any combination specified in the description,and does not necessarily preclude chemical interactions among theconstituents of a mixture once mixed; the first definition of an acronymor other abbreviation applies to all subsequent uses herein of the sameabbreviation and applies mutatis mutandis to normal grammaticalvariations of the initially defined abbreviation; and, unless expresslystated to the contrary, measurement of a property is determined by thesame technique as previously or later referenced for the same property.Unless stated to the contrary, all R groups include H, C₁₋₁₂ alkyl,C₁₋₁₂ alkynyl, C₁₋₁₂ alkenyl, C₁₋₁₂ fluoroalkyl, Cl, F, Br, nitro,cyano, or C₆₋₁₄ aryl, or C₅₋₁₄ hetereoaryl.

It is also to be understood that this invention is not limited to thespecific embodiments and methods described below, as specific componentsand/or conditions may, of course, vary. Furthermore, the terminologyused herein is used only for the purpose of describing particularembodiments of the present invention and is not intended to be limitingin any way.

It must also be noted that, as used in the specification and theappended claims, the singular form “a,” “an,” and “the” comprise pluralreferents unless the context clearly indicates otherwise. For example,reference to a component in the singular is intended to comprise aplurality of components.

Throughout this application, where publications are referenced, thedisclosures of these publications in their entireties are herebyincorporated by reference into this application to more fully describethe state of the art to which this invention pertains.

The term “liposome” as used herein refers to artificially preparesmembrane enclosed vesicle composed of bilayers of amphiphiles, which arecharacterized by having a hydrophilic and a hydrophobic group on thesame molecule. Liposomes include one to several of these bilayers.

The term “alkyl”, as used herein, unless otherwise indicated, includesC₁₂ saturated monovalent hydrocarbon radicals having straight orbranched moieties, including, but not limited to, methyl, ethyl,n-propyl, iso-propyl, n-butyl, sec-butyl, tert-butyl, and the like.

The term “alkenyl”, as used herein, unless otherwise indicated, includesC₂_alkyl groups, as defined above, having at least one carbon-carbondouble bond, such as CH₂—CH═CH₂.

The term “alkynyl”, as used herein, unless otherwise indicated, includesC₂_₂ alkyl groups, as defined above, having at least one carbon-carbontriple bond, such as —CH₂C—CH.

The term “alkylenyl”, as used herein, unless otherwise indicated,includes C₁₋₁₂ saturated divalent hydrocarbon radicals having straightor branched moieties.

The term “cryptand” as used herein mean a bi- and polycyclicpolyazo-polyether multi-dentate ligand, where three-coordinate nitrogenatoms provide the vertices of a three-dimensional structure.

The term “thiacryptand” as used herein mean a cryptand with at least oneoxygen atom replaced by a sulfur atom.

The term “carboxylated” as used herein means that a chemical moiety issubstituted with CO₂H (or CO₂).

The term “subject” refers to a human or animal, including all mammalssuch as primates (particularly higher primates), sheep, dog, rodents(e.g., mouse or rat), guinea pig, goat, pig, cat, rabbit, and cow.

In an embodiment, a composition useful for magnetic resonance imagingapplications is provided. The composition includes a plurality ofliposomes, europium (Eu) metal complexes disposed within the liposomes.Each europium metal complex including a europium metal ion and a firstmulti-dentate ligand selected from the group consisting of cryptands andthiacryptands. The composition also includes if necessary counter ionsto maintain charge neutrality (e.g., one or more counter-ions thatbalance the charge of the europium metal ion and the multi-dentateligand). The europium metal atom typically has a formal charge of +2 or+3 depending on the oxidation state. Depending on the substitutes on themulti-dentate ligand, the ligand may also have a formal charge underphysiological conditions. For example, carboxylates may have a negativecharge under physiological conditions. The counter-ions render theeuropium metal complex neutral. Examples of counter-ions when thecombination of europium metal atom and multi-dentate ligand are positiveinclude, but are not limited to, halide, hydroxide, bicarbonate,phosphate, and the like. Examples of counter-ions when the combinationof europium metal atom and multi-dentate ligand are negative include,but are not limited to, alkali metal ions (e.g., Na⁺, K⁺), H⁺, othermetal ions, and the like. FIG. 1 illustrates a liposome 10 encapsulatingeuropium metal complex 12. Surprisingly, the europium metal ions areswitchable between a 2+ and 3+ oxidation state even when encapsulated inthe liposome as illustrated in FIG. 1. The oxidation of Eu²⁺ to Eu³⁺provides orthogonal modes of detection by MRI. The Eu^(2+/3+) oxidationstate switch offers an ideal platform for oxygen-responsive contrastenhancement. In the variations and refinements set forth below, changesto ligand structure made the corresponding oxidation potential of Eu²⁺tunable over a physiologically relevant range. For example, FIG. 2provides the oxidation potential for several complexes therebydemonstrating the tenability and stability of these compounds. Theencapsulation of the europium metal complexes in liposomesadvantageously provides concentration-independent diagnostic imaging ofredox-active disease states using the chemistry of Eu.

Typically, each liposome includes a phospholipid such as fatty aciddi-esters of phosphatidylcholine, ethylphosphatidylcholine,phosphatidylglycerol, phosphatidic acid, phosphatidylethanolamine,phosphatidylserine or sphingomyelin. A particularly useful phospholipidis 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine. In many usefulvariations, the composition also includes a pharmaceutically acceptableaqueous carrier such as an aqueous buffer. In a refinement, theliposomes have an average diameter less than about 200 nm. In stillother refinements, the liposomes have an average diameter less thanabout, in increasing order of preference, 200 nm, 150 nm, 130 nm, 120nm, and 100 nm. In yet other refinements, the liposomes have an averagediameter greater than about, in increasing order of preference, 40 nm,50 nm, 60 nm, 70 nm, and 80 nm.

The europium metal complex may be included in the liposomes by any ofthe methods known to those skilled in the art for including drugs intoliposomes. Examples of such techniques are set forth in J. S. Dua etal., Liposome: Methods of Preparation and Applications, InternationalJournal of Pharmaceutical Studies and Research, Vol. 111/Issue11/April-June (2012), p. 14-20; the entire disclosure of which is herebyincorporated by reference. In general, an aqueous solution of theeuropium metal complex set forth above is combined with a liposomeforming precursor or a preformed liposome resulting in inclusion of thecomplex in the cavity of the liposome. Specific methods for forming theliposomes and incorporating the europium metal complex into the liposomeinclude, but are not limit to, the thin-film hydration method, the etherinjection method, ethanol injection method, detergent removal method,and the like. In a refinement, the liposomes are unilamellar. In anotherrefinement, the liposomes are multilamellar. In some refinements, theliposomes further include additional amphiphilic compound such ascholesterol.

In a variation, the multi-dentate ligand is a substituted2.2.2-Cryptand, an unsubstituted 2.2.2-Cryptand, a substituted2.2.2-thiacryptand, or an unsubstituted 2.2.2-thiacryptand. It should bepointed out that the europium metal complexes used herein areoxidatively stable in aqueous medium with the degree of stabilizationbeing dependent on the substituents on the cryptand. In this variation,the multi-dentate ligand is generally described by formula I:

wherein:Y¹, Y², Y³, Y⁴, Y⁵ and Y⁶ are each independently O or S;R₁, R₂, R₃ are each independently H, C₁₋₁₂ alkyl, C₁₋₁₂ alkynyl, C₁₋₁₂alkenyl, C₁₋₁₂ fluoroalkyl, Cl, F, Br, nitro, cyano, or C₆₋₁₄ aryl,C₅₋₁₄ hetereoaryl, or 5 and 6 membered rings formed by combining R₁ onadjacent carbon atoms or R₂ and R₃ on adjacent carbon atoms, ═O bycombining R₁, R₂, or R₃ on the same carbon atom, ═S by combining R₁, R₂,or R₃ on the same carbon atom, or ═NR by combining R₁, R₂, or R₃ on thesame carbon atom; andR is H or C₁₋₁₂ alkyl. It should be appreciated that in accordance withthis terminology the R₁ may be different from each other, the R₂ may bedifferent from each other, and the R₃ may be different from each other.In a refinement, R₁ on adjacent carbon atoms or R₂ and R₃ on adjacentcarbon atoms form a phenyl group. In another refinement, R₁, R₂, or R₃are each independently H, phenyl, or biphenyl. In some refinements, R₂and R₃ are hydrogen and one of the R₁ is not hydrogen. In otherrefinements, R₂ and R₃ are hydrogen and two of the R₁ are not hydrogen.Examples of cryptands and thiacryptands and europium complexes includingthese moieties that useful in the compositions of the present inventionare set forth in U.S. Pat. Pub. No. 20130078189 and in J. Garcia et al.,Physical Properties of Eu ²⁺-Containing Cryptates as Contrast Agents forUltrahigh-Field Magnetic Resonance Imaging, Eur. J. Inorg. Chem. 2012,2135-2140; the entire disclosures of which are hereby incorporated byreference in their entirety. FIG. 3 provides specific examples ofcryptands and thiacryptands that are useful as multi-dentate ligands inthe present invention. FIGS. 4 and 5 provide synthetic pathways forpreparing some of the Eu complexes of the present embodiment. Thefollowing formula provides a representation of the Eu complex with theligand of formula I:

where c is the charge of the combination of the europium metal atom andthe multi-dentate ligand. In a refinement, c is from −3 to +3. When themulti-dentate ligand is neutral, c is 2 or 3. Xp represents a number ofcounter-ions necessary for charge neutrality. X is a counter-ion as setforth above and p is the number of counter-ion necessary for chargeneutrality. Typically, p is an integer from 0 to 3.

In a variation of the present embodiment, the composition furtherincludes an antioxidant. The inclusion of an antioxidant limits thetoxicity of europium metal complexes and improves the stability (i.e.,oxidative stability). Both in vitro (T₁-based assays in the air) and invivo (maximum tolerated dose assays coupled with biodistributionstudies) studies can be used to evaluate the toxicity of the complexes.Examples of antioxidants include, but are not limited to, glutathione,2-mercaptoethanol, dithiothreitol, S-adenosylmethionine,dithiocarbamate, dimethylsulfoxide, cysteine, methionine, cysteamine,oxo-thiazolidine-carboxylate, timonacic acid, malotilate, 1,2-dithiol3-thione, 1,3-dithiol 2-thione, lipoamide, sulfarlem, oltipraz, taurine,N-acetylcysteine, and combinations thereof.

Additional examples of the multi-dentate ligand are described by thefollowing formulae:

wherein Y, Z, Y¹ and Y² are each independently O or S; X¹, X², X³ areindependently nitro, Cl, F, Br, I, CH₃, NH₂, or CH₂CO₂H; and R, R′ areindependently nitro, Cl, F, Br, I, CH₃, NH₂, or CH₂CO₂H.

Another example of the multi-dentate ligand has the following formula:

wherein x, y, and z are 0, 1, 2, or 3 with the proviso that the sum ofx, y, and z is 3.

Another example of the multi-dentate ligand has the following formula:

wherein x and y are 0, 1, 2, or 3 with the proviso that the sum of x andy is 3.

In another embodiment, a compound useful as an MRI contrast agent havingformula II is provided:

wherein:n, o are each independently 1 or 2;L₁ is a multi-dentate ligand that bonds to Eu;L₂ is a multi-dentate ligand that bonds to Gd;X is a counter-ion as set forth above and p is the number of counter-ionnecessary for charge neutrality. Typically, p is an integer from 0 to 3;andSp is a spacer moiety that provides separation between Eu and Gd.Typically, Sp includes —(CH₂)_(n)—, C₂₋₁₂ alkylenyl, C₂₋₁₂ alkynyl,C₆₋₁₂ aryl, C₅₋₁₂ heteroaryl, urea groups (—NHCO—NH—), thiourea groups(—NHCSNH—), carbamate groups (—NHCOO—), C₁₋₁₀ amine groups, OH groups,NH groups C₁₋₁₀ alkenyl groups, and combinations thereof where n is 1 to12. In a refinement, the compound having formula (II) is disposed with aliposome as set forth above.Examples of Sp include:

—(CH₂)_(n)—HNCONH—, —(CH₂)_(n)—HNCSNH—, and —(CH₂)_(n)—. In a variation,L₁ is described by the following formula:

with at least one R₁, R₂, or R₃ being a bond to Sp or having a bond toSp. Y¹, Y², Y³, Y⁴, Y⁵, and Y⁶ are each independently O or S; R₁, R₂, R₃are each independently H, C₁₋₁₂ alkyl, C₁₋₁₂ fluoroalkyl, Cl, F, Br,nitro, cyano, or C₆₋₁₄ aryl, C₅₋₁₄ hetereoaryl, or 5 and 6 memberedrings formed by combining R₁ on adjacent carbon atoms or R₂ and R₃ onadjacent carbon atoms, ═O by combining R₁, R₂, or R₃ on the same carbonatom, ═S by combining R₁, R₂, or R₃ on the same carbon atom, or ═NR bycombining R₁, R₂, or R₃ on the same carbon atom; and R is H or C₁₋₁₂alkyl. In a refinement, R₁ on adjacent carbon atoms or R₂ and R₃ onadjacent carbon atoms form a phenyl group. In another refinement, R₁,R₂, R₃ are each independently H, phenyl, or biphenyl. In a variation, L₂include a plurality of groups having formula IV:

In a refinement, L₂ includes a moiety having the following formula:

Specific examples of compounds having formula (II) are the followingcompounds:

It should be appreciated that the compounds of the present embodimentare capable of functioning as “universal field” agents that works atboth low field and high field instruments.

In another embodiment, a europium metal complex comprising a europiummetal ion, a multi-dentate having formula VIII, and if necessarycounter-ions to maintain charge neutrality is provided:

The europium metal complex is represented by the following formula:

wherein:c is the charge of the combination of the europium metal atom and themulti-dentate ligand. In a refinement, c is from −3 to +3. When themulti-dentate ligand is neutral, c is 2 or 3. Xp represents a number ofcounter-ions necessary for charge neutrality. X is a counter-ion as setforth above and p is the number of counter-ion necessary for chargeneutrality. Typically, p is an integer from 0 to 3;Y¹, Y², Y³, Y⁴, Y⁵ and Y⁶ are each independently O or S;R₁, R₂, R₃ are each independently H, C₁₋₁₂ alkyl, C₁₋₁₂ alkynyl, C₁₋₁₂alkenyl, C₁₋₁₂ fluoroalkyl, Cl, F, Br, nitro, cyano, or C₆₋₁₄ aryl,C₅₋₁₄ hetereoaryl, or 5 and 6 membered rings formed by combining R₁ onadjacent carbon atoms or R₂ and R₃ on adjacent carbon atoms, ═O bycombining R₁, R₂, or R₃ on the same carbon atom, ═S by combining R₁, R₂,or R₃ on the same carbon atom, or ═NR by combining R₁, R₂, or R₃ on thesame carbon atom;R is H or C₁₋₁₂ alkyl. In a refinement, R₁, R₂, or R₃ on adjacent carbonatoms or R₂ and R₃ on adjacent carbon atoms form a phenyl group. Inanother refinement, R₁, R₂, or R₃ are each independently H, phenyl, orbiphenyl; andR₄, R₅, R₆, R₇ are each independently hydrogen, cyano, nitro, Cl, F, Br,I, CH₃, NH₂, C₆₋₁₅ aryl, carboxylated C₆₋₁₈ aryl, C₅₋₁₅ heteroaryl,carboxylated C₅₋₁₈ heteroaryl, C₁₋₁₂ alkyl, phenyl, carboxylated phenyl,carboxylated C₂₋₁₂ alkyl, —CO₂H, —CH₂CO₂H,

R₈, R₉, R₁₀, R₁₁, R₁₂ are each independently H or CO₂H. In should beappreciated that these compounds can be encapsulated in liposomes as setforth above. Refinements of the present embodiment are useful as“ultra-high field” T1 imaging agents.

In another embodiment, a europium metal complex comprising a europiummetal ion, a multi-dentate having formula IX, and if necessarycounter-ions to maintain charge neutrality is provided:

The europium metal complex is represented by the following formula:

wherein:c is the charge of the combination of the europium metal atom and themulti-dentate ligand. In a refinement, c is from −3 to +3. When themulti-dentate ligand is neutral, c is 2 or 3. Xp represents a number ofcounter-ions necessary for charge neutrality. X is a counter-ion as setforth above and p is the number of counter-ion necessary for chargeneutrality. Typically, p is an integer from 0 to 3;Y¹, Y², Y³, Y⁴, Y⁵, and Y⁶ are each independently O or S;R₁, R₂, R₃ are each independently H, C₁₋₁₂ alkyl, C₁₋₁₂ alkynyl, C₁₋₁₂alkenyl, C₁₋₁₂ fluoroalkyl, Cl, F, Br, nitro, cyano, or C₆₋₁₄ aryl,C₅₋₁₄ hetereoaryl, or 5 and 6 membered rings formed by R₁ on adjacentcarbon atoms or R₂ and R₃ on adjacent carbon atoms, ═O by combining R₁,R₂, or R₃ on the same carbon atom, ═S by combining R₁, R₂, or R₃ on thesame carbon atom, or ═NR by combining R₁, R₂, or R₃ on the same carbonatom; andR is H or C₁₋₁₂ alkyl. In a refinement, R₁ on adjacent carbon atoms orR₂ and R₃ on adjacent carbon atoms form a phenyl group. In anotherrefinement, R₁, R₂, or R₃ are each independently H, phenyl, or biphenyl.In should be appreciated that these compounds can be encapsulated inliposomes as set forth above.

In another embodiment, a europium metal complex comprising a europiummetal ion, a multi-dentate having formula X, and if necessarycounter-ions to maintain charge neutrality is provided:

The europium metal complex is represented by the following formula:

wherein:c is the charge of the combination of the europium metal atom and themulti-dentate ligand. In a refinement, c is from −3 to +3. When themulti-dentate ligand is neutral, c is 2 or 3. Xp represents a number ofcounter-ions necessary for charge neutrality. X is a counter-ion as setforth above and p is the number of counter-ion necessary for chargeneutrality. Typically, p is an integer from 0 to 3;Y¹, Y², Y³, Y⁴, Y⁵, and Y⁶ are each independently O or S;R₁, R₂, R₃ are each independently H, C₁₋₁₂ alkyl, C₁₋₁₂ alkynyl, C₁₋₁₂alkenyl, C₁₋₁₂ fluoroalkyl, Cl, F, Br, nitro, cyano, or C₆₋₁₄ aryl,C₅₋₁₄ hetereoaryl, or 5 and 6 membered rings formed by combining R₁ onadjacent carbon atoms or R₂ and R₃ on adjacent carbon atoms, —O bycombining R₁, R₂, or R₃ on the same carbon atom, —S by combining R₁, R₂,or R₃ on the same carbon atom, or —NR by combining R₁, R₂, or R₃ on thesame carbon atom; andR is H or C₁₋₁₂ alkyl. In a refinement, R₁ on adjacent carbon atoms orR₂ and R₃ on adjacent carbon atoms form a phenyl group. In anotherrefinement, R₁, R₂, or R₃ are each independently H, phenyl, or biphenyl.In should be appreciated that these compounds can be encapsulated inliposomes as set forth above.

In another embodiment, a method of magnetic resonance imaging of anorgan or organ structure in a subject. Eu²⁺-based complexes encapsulatedinside liposomes can be detected with conventional T₁-weighted MRimaging while oxidation to Eu³⁺ will result in a contrast agent that canbe detected with paramagnetic chemical exchange saturation transfer(PARACEST) imaging. The method includes a step of administering aliposome composition to the subject and then taking images of the organof interest in the subject by magnetic resonance imaging. The liposomecomposition including a liposome; and a europium metal complex disposedwithin the liposome. The europium metal complex includes a europiummetal ion and a first multi-dentate ligand selected from the groupconsisting of cryptands and thiacryptands, and one or more counter-ionsthat balances the charge of the europium metal ion. The details of theliposome, europium metal complex, and the multi-dentate ligands are setforth above. In one refinement, the magnetic resonance imaging isT₁-weighted imaging. In another refinement, the magnetic resonanceimaging is by chemical exchange saturation transfer (CEST) and inparticular, PARACEST. In still another refinement, the method tracks themigration of the contrast agent with T₁-weighted imaging and then upondisappearance of T₁ enhancement, the imaging mode of detection ischanged to CEST. In this these refinements, the presence of CEST wouldindicate oxidation, and an absence of CEST enhancement would indicateclearance of the contrast agent. Furthermore, CEST enhancement could beused to indicate one or more specific disease states because theoxidation potential, and consequently loss of T₁ enhancement, of Eu² istunable through ligand structure modifications. In general, the magneticresonance imaging involves positioning the subject in a magnetic fieldtypically having a spatial gradient. AN electromagnetic pulse (i.e.,radiofrequency pulse) is applied to the subject with the frequency beingvaried over a region where hydrogen atoms resonate. At the resonancefrequency for the water hydrogen atoms in the spatial region beingscanned, absorption occurs with the signal subsequently emitted beingmonitored to provide the T₁-weighted image. In CEST imaging, thehydrogen atoms for water in the liposomes are brought to saturation bythe applied electromagnetic pulse. The resulting magnetization istransferred to mobile water molecules outside of the liposomes with theemission from the hydrogen atoms on these mobile water molecules beingmonitored to provide the data required for CEST imaging.

The Examples below are included to demonstrate preferred embodiments ofthe invention. It should be appreciated by those of skill in the artthat the techniques disclosed in the Examples represent techniques andcompositions discovered by the inventors to function well in thepractice of embodiments disclosed herein, and thus can be considered toconstitute preferred modes for its practice. However, those of skill inthe art should, in light of the present disclosure, appreciate that manychanges can be made in the specific embodiments which are disclosed andobtain a like or similar result without departing from the spirit andscope of embodiments disclosed herein.

The compositions set forth above use liposomes because their aqueousinner cavity can encapsulate water-soluble contrast agents to improvethe sensitivity of CEST by increasing the ratio of chemically shiftedwater protons (inside liposomes) to bulk water protons (outsideliposomes) (Aime, S.; Castelli, D. D.; Terreno, E. Angew. Chem. Int. Ed.2005, 44, 5513-5515). Liposome composition was adapted from a reportthat used 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine andcholesterol (Gianolio, E.; Porto, S.; Napolitano, R.; Baroni, S.;Giovenzana, G. B.; Aime, S. Inorg. Chem. 2012, 51, 7210-7217), andliposomes were characterized using dynamic light scattering. The averagediameter measured before and after air exposure was 110±7 and 106 t 7nm, respectively, where the error is the standard error calculated fromthe average polydispersity index values. The average liposomepolydispersity index value before and after air exposure was 0.14±0.01and 0.10±0.06, respectively, where the polydispersity index error is thestandard error at the 95% confidence interval. These size distributiondata indicate that average liposome size was not different before andafter oxidation (Student t test) and, consequently, not affected by theintraliposomal formation of Eu³.

To evaluate the response of our liposomes, suspensions of liposomescontaining Eu(2.2.2)²⁺ were measured before and after exposure to airand observed T₁ lengthening upon air exposure (0.4 and 2.8 s before andafter air exposure, respectively, 24° C., 11.7 T, 45 mM Eu). Thisobservation is in good agreement with the T₁-shortening nature ofEu(2.2.2)²⁺ (Garcia, J.; Neelavalli, J.; Haacke, E. M.; Allen, M. J.Chem. Commun. 2011, 47, 12858-12860 and Garcia, J.; Kuda-Wedagedara, A.N. W.; Allen, M. J. Eur. J. Inorg. Chem. 2012, 2012, 2135-2140) andindicates that oxidation to form Eu³ caused the observed lengthening ofT₁. Furthermore, the data suggest the water-exchange rate betweenintraliposomal and bulk water is sufficiently fast for positive contrastenhancement.

To characterize the dual-mode behavior of Eu-containing liposomes,response were measured before and after air exposure using in vitroimage intensities as a function of frequency offset of presaturation at7 T. The intensity data was fitted with a Lorentzian function usingleast squares fitting to reference bulk water to 0 ppm (FIG. 6).Lorentzian fitting was used because the sample images were acquiredsimultaneously and the bulk water signals were not centered at 0 ppmlikely due to inhomogeneities in the magnetic field. Interestingly,saturation of the bulk water signal after air exposure was not observed.The CEST spectrum revealed that liposomes before and after 24 hour airexposure had exchangeable intraliposomal water protons at 1.4 and 1.2ppm, respectively, relative to bulk water. A possible explanation forthe observation of CEST both before and after oxidation is that adifference in osmolality between intraliposomal and bulk water causedosmotic shrinking of liposomes. Intraliposomal osmolality influences thechemical shift of intraliposomal water protons, and this phenomenon hasbeen demonstrated for Gd³⁺-containing liposomes.

A magnetization transfer ratio asymmetry analysis (MTR_(asym), FIG. 7)was performed because the exchangeable signals appeared partiallyoverlapped with the bulk water signal. MTR_(asym) analysis removes theeffect of direct saturation of bulk water to reveal asymmetries in theCEST spectrum. MTR_(asym) values were calculated from the Lorentzianfitting using (S^(Δω)−S^(Δω))/S₀, where S^(−Δω) and S^(Δω) are the bulkwater signal intensities at the negative and positive frequency offsets,respectively, from the bulk proton frequency referenced to 0 ppm; and S₀is the bulk water signal intensity after presaturation at −5 ppm. TheMTR_(asym) spectrum confirmed the existence of exchangeable pools ofintraliposomal water protons and also revealed increased CESTenhancement for liposomes after air exposure. One explanation forincreased CEST after air exposure is that the presence of Eu³⁺chemically shifted intraliposomal water protons and added to the CESTenhancement. Another possible explanation is that oxidation to Eu³⁺decreased intraliposomal osmolality, which shrunk the liposomes andincreased CEST intensity. This explanation is consistent with ourobservations because shrinking of liposomes through an osmotic pressuregradient is not easily detected by dynamic light scattering. The latterexplanation is based on the kinetic stability of Eu(2.2.2)³⁺ being lowerthan that of Eu(2.2.2)²⁺. Accordingly, Eu³⁺ is likely present as amixture of species within the liposomes including Eu(2.2.2)³⁺, Eu³⁺aqua, and phosphate complexes. A third possible explanation for thechange in observed CEST is that the presence of multiple Eu³⁺-containingspecies altered the water-permeability of the liposome membrane toincrease CEST enhancement after air exposure. Investigations exploringthese three possibilities, which might also explain the incompletesaturation of bulk water after air exposure, are currently underway.

To visualize the nature of the Eu^(2+/3+) responses, in vitro images ofsuspensions of the liposomes were acquired before and after exposure toair (FIG. 8). The T₁-weighted images confirmed positive contrastenhancement for the Eu(2.2.2)²⁺-containing liposomes and also revealedno difference in signal intensity between water and the oxidizedEu³⁺-containing liposomes at the 95% confidence interval (Student ttest). To quantify the CEST enhancement, the phantom image intensitieswere used to calculate % CEST defined as (1−M_(on)/M_(off))×100, whereM_(on) and M_(off) are the average signal intensities at the on- andoff-resonance positions. The CEST map confirmed the presence ofexchangeable intraliposomal water before and after oxidation and that %CEST increased from 52% to 77% after air exposure. These datademonstrate a distinct dual-mode response and reveal the oxidation stateof Eu without knowledge of its concentration. With this demonstration ofdistinct orthogonal imaging, we envision tracking the migration of thecontrast agent with T₁-weighted imaging. Upon disappearance of T₁enhancement, the imaging mode of detection would be changed to CEST. Thepresence of CEST would indicate oxidation, and an absence of CESTenhancement would indicate clearance of the contrast agent. Furthermore,CEST enhancement could be used to indicate one or more specific diseasestates because the oxidation potential, and consequently loss of T₁enhancement, of Eu²⁺ is tunable through ligand structure modifications.Accordingly, our in vitro data provide a strong framework for optimizingour system for in vivo imaging.

To demonstrate that the liposomes did not leach Eu, the oxidizedliposomes were filtered, and the Eu concentration of the filtrate wasmeasured to be below the detection limit (<66 nM) of inductively coupledplasma optical emission spectroscopy.

In conclusion, embodiments of the invention provide the firstoxidation-responsive dual-mode contrast agent for MRI based on the redoxchemistry of Eu. Contrast enhancement in orthogonal imaging modes allowsfor the detection of Eu oxidation states without knowledge of contrastagent concentration. For these reasons, we expect this system to openthe door for molecular imaging using the Eu^(2+/3+) redox switch.

Experimental Procedures

Commercially available chemicals were of reagent-grade purity or betterand were used without further purification unless otherwise noted. Waterwas purified using a PURELAB Ultra Mk2 water purification system (ELGA)and degassed prior to use. NMR spectroscopy and inductively coupledplasma optical emission spectroscopy (ICP-OES) analyses were performedat the Lumigen Instrument Center in the Department of Chemistry at WayneState University. In vitro phantom imaging was performed at Henry FordHospital.

Inversion-recovery T₁ measurements were obtained using a Varian VNMRS500 (499.48 MHz, 11.7 T) spectrometer before air exposure or after 24 hof air exposure. Deuterium oxide (300 mOsm NaCl) was added to makeliposome suspensions 5% D₂O (v/v) for the purpose of locking andshimming.

MRI scans were performed with a 7 T Varian small animal MRI scanner(299.44 MHz, 7.0 T) equipped with a 12 cm bore magnet and a 38 mmdiameter homemade transmit/receive quadrature birdcage coil. Samplesincluded liposomes that were not exposed to air, liposomes that wereexposed to air for 24 h, and water. The T₁-weighted images were acquiredat ambient temperature (echo time: 11 ms; repetition time: 320 ms; sevenimage slices at 1 mm thickness; 24×24 mm² field of view; and fouraverages). The liposome-encapsulated Eu³⁺ (chemical exchange saturationtransfer, CEST) effects were measured at ambient temperature under thesame parameters used in a previous CEST MRI study. A RARE MRI pulsesequence with a RARE factor of 8 (repetition time/echo time, 4.0 s/11ms) was applied with a 17 T saturation power for 2 s. A total of 64 swas required to acquire a single MR image with 128×128 pixels thatcovered a 24×24 mm² field of view, a single slice with a thickness of 1mm, and a single average. The water signal was measured for each phantomwhen saturation was applied between 5 and −5 ppm in 0.2 ppm incrementsto measure the CEST effect of liposomes, and FIG. 4 in the manuscriptwas acquired at 1.2 ppm (S^(Δω)) and −1.2 ppm (S^(−Δω)).

Varian flexible data format (FDF) files were converted to tagged imagefile format (TIFF) files with a MATLAB code. TIFF files were processedto produce chemical exchange saturation transfer (CEST) spectra bymeasuring pixel intensities with ImageJ 1.47. A magnetization transferratio asymmetry analysis (MTR_(asym)) of the Lorentzian fit wasperformed because the exchangeable intraliposomal proton signalpartially overlapped with the bulk water proton signal. MTR_(asym) wascalculated using eq 1.4

$\begin{matrix}{{MTR}_{asym} = {\frac{S^{- {\Delta\omega}} - S^{- {\Delta\omega}}}{S_{0}}.}} & {{eq}\mspace{14mu} 1}\end{matrix}$

In eq 1, S−^(Δω) and S^(Δω) are the bulk water signal intensities at thenegative and positive frequency offsets, respectively, from the bulkwater proton frequency referenced to 0.00 ppm; and S₀ is the bulk watersignal intensity after presaturation at −5 ppm. Percent CEST (% CEST)was calculated using eq 2.5

$\begin{matrix}{{\%{CEST}} = {\left( {1 - \frac{M_{on}}{M_{off}}} \right)100.}} & {{eq}\mspace{20mu} 2}\end{matrix}$

In eq 2, Mon and Moff are the average signal intensities (calculatedwith ImageJ) of the same phantom tube slice at 360 Hz (1.2 ppm) and −360Hz (−1.2 ppm), respectively. The CEST image was created by subtractingthe TIFF slice at 360 Hz (1.2 ppm) from the identical slice at −360 Hz(−1.2 ppm) and the difference was divided by the slice at 360 Hz (−1.2ppm). The % CEST scale bar was created by calibrating the pixel range ofthe CEST image to the maximum % CEST value obtained from eq 2 using alinear fit.

Dynamic light scattering data were obtained using a Malvern ZetasizerNano-ZS instrument (ZEN3600) operating with a 633 nm wavelength laser.Dust was removed from samples by filtering through 0.2 μm hydrophilicfilters (Millex-LG, SLLGR04NL). Liposome samples were prepared for lightscattering experiments by diluting (1:10) purified liposome suspensionsin iso-osmolar phosphate-buffered saline [PBS; Na₂HPO₄ (29 mM), NaH₂PO₄(46 mM), NaCl (57 mM), and KCl (2.1 mM); pH 7.0]. For liposome sizemeasurements with no air exposure, air-tight cuvettes were filled in aglovebox under an atmosphere of Ar.

ICP-OES measurements were acquired on a Jobin Yvon Horiba Ultimaspectrometer. All samples were diluted with 2% HNO₃, which was also usedfor blank samples during calibration. The calibration curve was createdusing the Eu emission intensity at 381.965 nm for a 1-10 ppmconcentration range (diluted from Alfa Aesar Specpure AAS standardsolution, Eu₂O₃ in 5% HNO₃, 1000 μg/mL), and all samples were diluted tofall within this range.

Preparation of Hydration Solution

The hydration solution was prepared by stirring an aqueous solution ofEuCl₂ and 4,7,13,16,21,24-hexaoxa-1,10-diazabicyclo[8.8.8]hexacosane(cryptand) for 12 h under an atmosphere of Ar followed by aphosphate-buffer-workup.⁶ To account for loss of phosphate during theprecipitation step of this experiment, a PBS stock solution was preparedwith a high concentration of phosphate (1 M). The purpose of the highphosphate concentration was to ensure PBS buffer capacity was not lostupon phosphate precipitation in the presence of uncomplexed Eu³⁺ in theoxygen-exposed samples and to maintain physiological osmolality (300mOsm). This PBS solution was prepared in a glovebox under an atmosphereof Ar by dissolving anhydrous dibasic sodium phosphate (42.6 g, 0.300mol), monobasic sodium phosphate monohydrate (27.6 g, 0.200 mol), sodiumchloride (22.3 g, 0.381 mol), and potassium chloride (1.01 g, 13.6 mmol)in H₂O (500 mL). The pH of the resulting solution was brought to 7.0with the addition of solid sodium hydroxide (3.87 g, 96.8 mmol).

To a 4 mL glass vial equipped with a magnetic stir bar was added aqueousEuCl₂ (261.4 μL, 206.6 mM, 1 equiv) and aqueous cryptand (207.7 μL,260.0 mM, 1 equiv) under an atmosphere of Ar. The resulting clear,colorless solution was stirred for 12 h before addition of PBS [60 μL;Na₂HPO₄ (383 mM), NaH₂PO₄ (617 mM), NaCl (762 mM), KCl (27.2 mM); pH7.0] and water (670.9 μL) to bring the total volume to 1.20 mL. Uponaddition of PBS, a slightly turbid suspension formed that was stirredfor 1 h and then filtered through a 0.2 μm hydrophilic filter. The Euconcentration of the clear, colorless filtrate was determined byICP-OES. This filtrate was used for liposome preparation.

Preparation of Liposomes

Liposomes were prepared via the thin-film hydration technique (Liu, G.;Liang, Y.; Bar-Shir, A.; Chan, K. W. Y.; Galpoththawela, C. S.; Bernard,S. M.; Tse, T.; Yadav, N. N.; Walezak, P.; McMahon, M. T.; Bulte, J. W.M.; van Zijl, P. C. M.; Gilad, A. A. J. Am. Chem. Soc. 2011, 133,16326-16329). To a 4 mL vial was added1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (22.0 mg, 2.89 μmol,1.4 equiv), cholesterol (8.0 mg, 2.1 μmol, 1 equiv), and chloroform (1mL) to produce a clear, colorless solution. Solvent was removed underreduced pressure to afford a visible film on the bottom of the vial.Under an atmosphere of Ar, the hydration solution (1.15 mL) and vialcontaining the lipid thin film were placed in a water bath at 55° C. for30 min, and then the hydration solution was added to the vial containingthe thin film. The resulting white suspension was stirred at 55° C. for1 h. Extrusion of the suspension was accomplished using a mini-extruderand heating block (Avanti Polar Lipids, Alabaster, Ala., USA) heated to55° C. (4 passes through a 0.2 μm polycarbonate filter followed by 15passes through a 0.1 μm polycarbonate filter). After extrusion, thesuspension was allowed to cool to ambient temperature within theAr-filled glovebox for 1 h.

Non-encapsulated Eu²-containing cryptate was removed from the liposomesuspension in an Ar-filled glovebox via spin filtering (Amicon Ultraregenerated cellulose 3,000 molecular weight cut off). The liposomesuspension was filtered in aliquots because the volume of the suspensionexceeded the volume of the spin filter. When the volume of suspension inthe filter reached 0.3 mL after spinning, the volume was brought to 0.5mL with the addition of iso-osmolar (300 mOsm) PBS prepared by dilutionof the PBS solution described above. Spin-filtered fractions werecollected until Eu was not detectable by ICP-OES (14 fractions).

FIG. 9 shows a Plot of T₁ vs time of the EuII-containing complex ofligand 4 (3 mM) in the absence (□) and presence (∘) of glutathione (325mM). Samples were prepared in a glovebox in the absence of molecularoxygen and then poured into an NMR tube in the air outside of theglovebox at time=0. Pouring allowed samples to become aerated. Sampleswere kept at 37° C. and exposed to air for the duration of theexperiment. Oxidation of Eu² to Eu³ results in an increase in the T₁ ofthe solution. Error bars represent the standard error of the mean ofindependently prepared samples. This data demonstrates that theantioxidant glutathione increases the oxidation half-life of theEuII-containing complex of ligand 4 by approximately 4 hours.

These results demonstrate in vivo oxidative stability and supportdeveloping positive contrast agents for ultra-high field strength MRIusing Eu²⁺-based complexes. These probes, based on the stabilization ofEu²⁺, will be significant because they are expected to enable the use ofultra-high field strengths for imaging studies that are routinelycarried out at 1.5 and 3 T, as well as new studies that require bothhigh resolution imaging and contrast enhancement. In particular,compositions of the invention are useful at magnetic fields greater than3 T (e.g., 7 to 11 T). The present invention represents the first steptoward the use of the complexes as contrast agents at ultra-high fieldstrengths in preclinical research and diagnostic medicine. Subsequently,these improved contrast agents are expected to facilitate earlierdetection of disease when used in combination with higher field strengthMRI, and early diagnoses correspond to higher rates of successfultreatment. Furthermore, the Eu^(II)-based contrast agents will enablebetter monitoring of therapies, especially when both high resolution andcontrast enhancement are vital for success.

General Procedure for the Synthesis of E^(II)-Containing Cryptates fromLigands

A degassed aqueous solution of EuCl₂ (1 equiv) is mixed with a degassedaqueous solution of a cryptand (2 equiv). The resulting mixture isstirred for 12 hours at ambient temperature under Ar. Degassed PBS (10×)is added to make the entire reaction mixture 1× in PBS, and stirring iscontinued for 30 min. The concentration of Eu in the resulting solutionis verified by ICP-MS.

Multi-Dentate Ligand Having Formula VI:

GdCl₃.6H₂O (63.4 mg, 0.171 mmol) was dissolved in water (2 mL) and pHadjusted to pH 6 using NaOH. The metal solution was then added to asolution containing 1 (103 mg, 0.159 mmol) in water (4 mL), pH adjustedto 6 using NaOH. The reaction mixture was purged with argon and allowedto stir at ambient temperature for 24 h. Dialysis was performed using a100-500 Da membrane. The dialysis water was changed three times over thecourse of 10 h. The solution was then transferred from the dialysismembrane to a flask, and solvent was removed under reduced pressure. Acrude yield was then measured to be 134 mg, and some of this compound(5.2 mg, 0.0075 mmol) was dissolved in carbonate buffer (pH 9.8, 3 mL,0.5 M). The resulting solution was added to a solution containing theamine-substituted cryptand (LIGAND 36 from FIG. 3, 4.8 mg, 0.011 mmol)in the same carbonate buffer (2 mL). Throughout the combination of thetwo solutions, the pH was monitored via pH paper and kept between 9 and10. The reaction solution was allowed to stir at ambient temperature for11 h. Dialysis was performed using a 100-500 Da membrane, includingchanging of the dialysis water 3 times over the course of 24 h. Themixture was transferred to a flask and solvent was removed under reducedpressure where a resulting crude yield was measure to be 5.5 mg.High-performance liquid chromatography (HPLC) analysis of the resultingconjugation using a PFPP column showed elution between 3.156 and 3.174minutes using a 0-95% gradient (ramp began at 20 minutes, reached 95% at22 minutes, and decreased at 26 minutes and reached 0% again at 28minutes). This complex can be mixed with one equivalent of EuCl₂ toproduce the a europium metal complex.

5,6-(4-Fluorobenzo)-4,7,13,16,21,24-hexaoxa-1,10-diazabicyclo[8.8.8]hexacos-5-ane(Ligand 4 from FIG. 3)

A 2.0 g of 4-Fluorocatechole (1 equiv, 15.6 mmol) in 20 mL of acetonewas added to a solution of methyl bromo ester (3 equiv, 4.4 mL, 46.8mmol) and potassium carbonate (3 equiv, 6.5 g, 46.8 mmol) in 100 mL ofacetone for a period of 10 min under an Ar atmosphere while refluxing,and the resulting mixture was heated under reflux for 18 h under an Aratmosphere. After the reaction, excess K₂CO₃ was removed to yield a palered solution which was dried under reduced pressure, and the crude oilwas dissolved in ethyl acetate and washed three times with 15 mL ofwater. The resulting organic layer was dried under reduced pressure toyield 3.75 g of a pale yellow powdery solid (yield 88%). This material(3.57 g) of 4-fluorocatechole-o,o-diacetic acid methyl ester (13.1 mmol)and 1.43 g of Dowex50WX8-200 was added to a 120 mL of water and heatedunder reflux at 110° C. for 24 h under an Ar atmosphere withoutstirring. The resulting mixture was filtered, and the resin was washedwith MeOH. The filtrate was dried under reduced pressure to yield a paleyellow powdery solid, of which 0.40 g (1.8 mmol) was dissolved inthionyl chloride (5.0 mL, 68 mmol) under Ar and heated at reflux for 4h. Excess thionyl chloride was removed under reduced pressure, and theresidue was dissolved in anhydrous toluene (25 mL). The resultingsolution and a solution of 1,4,10,13-tetraoxa-7,16-diazacyclooctadecane(0.32 g, 1.2 mmol, 1.0 equiv) and triethylammine (0.50 mL, 3.3 mmol, 2.4equiv) in anhydrous toluene (25 mL) were added simultaneously (50 mL/h)to a separate flask containing anhydrous toluene (60 mL) at 0-5° C.under an Ar atmosphere. The solution was stirred for 12 h at ambienttemperature. An orange suspension formed was filtered, and the solventwas removed under reduced pressure to yield a brown solid. Purificationwas done using silica gel chromatography (9:1 CH₂Cl₂/methanol) andyielded 0.220 g (54%) of a fluffy yellow solid. The compound (0.10 g,0.21 mmoL, 1 equiv) was dissolved in 8.25 mL anhydrous THF, and to itwas added 6.5 mL of BH₃-THF, 1 M solution (6.4 mmoL, 30 equiv) slowlywhile stirring under Ar atmosphere at 0-5° C. When the addition wasdone, the solution was heated under reflux for 24 h. Then the solutionwas treated first with 6M HCl (8.25 mL) and second with water (8.25 mL)and heated under reflux for 6 h. The solution was then basified withNH₄OH and removed the solvent under reduced pressure. The salt wasfiltered out and dried again. Purification was done using silica gelchromatography (8:1 CH₂Cl₂/methanol) to yield 0.220 g (54%) of 1 a paleyellow solid.

5,6-(4-chlorobenzo)-4,7,13,16,21,24-hexaoxa-1,10-diazabicyclo[8.8.8]hexacos-5-ane(Ligand 32 from FIG. 3)

A solution of 0.924 g of 4-chlorocatechole (1 equiv, 6.92 mmol) in 10 mLof acetone was added to a solution of methyl bromo ester (3 equiv, 1.95mL, 20.76 mmol) and potassium carbonate (3 equiv, 2.827 g, 20 mmol) in50 mL of acetone for a period of 10 min under Ar at reflux, and theresulting mixture was heated under reflux for 30 h under an Aratmosphere. After the reaction, excess K₂CO₃ was filtered off and driedunder reduced pressure. Purification was done using silica gelchromatography (1:1 hexanes/ethyl acetate) yielded 1.800 g (98%) of ayellow solid. The resulting material (1.05 g, 3.46 mmol) and 0.541 g ofDowex50WX8-200 were added to 40 mL of water and heated under reflux for19 h under an Ar atmosphere without stirring. The resulting mixture wasfiltered and the resin was washed with MeOH. The filtrate was driedunder reduced pressure to yield a pale yellow powdery solid 0.93 g(98%). Some of this material (0.344 g, 1.31 mmol) was dissolved inthionyl chloride (5.0 mL, 68 mmol) under Ar and was heated at reflux for4 h. Excess thionyl chloride was removed under reduced pressure, and theresidue was dissolved in anhydrous toluene (20 mL). The resultingsolution and a solution of 1,4,10,13-tetraoxa-7,16-diazacyclooctadecane(0.340 g, 1.31 mmol, 1.0 equiv) and triethylammine (0.6 mL, 3.93 mmol,3.0 equiv) in anhydrous toluene (20 mL) were added simultaneously (50mL/h) to a separate flask containing anhydrous toluene (60 mL) at 0-5°C. under an Ar atmosphere. The solution was stirred for 16 h at ambienttemperature. An orange suspension formed and was filtered, and thesolvent was removed under reduced pressure to yield a yellow solid.Purification was done using silica gel chromatography (8:1CH₂Cl₂/methanol) to yield 0.380 g (60%) of a pale yellow oil. Thecompound (0.380 g, 0.78 mmoL, 1 equiv) was dissolved in 20 mL ofanhydrous THF and to it was added 20.0 mL of 1 M BH₃-THF (23.41 mmol, 30equiv) slowly while stirring under an Ar atmosphere at 0-5° C. When theaddition was done, the solution was warmed to room temperature andheated under refluxe for 30 h. Then the solution was treated first with6 M HCl (20 mL) and second with water (20 mL) and heated under refluxfor another 6 h. The solution was then basified with NH₄OH, and thesolvent was removed under reduced pressure. The salt was filtered outand dried again. Purification was done using silica gel chromatography(8:1 CH₂Cl₂/methanol) to yield 0.136 g (38%) of a white solid.

5,6-(4-bromobenzo)-4,7,13,16,21,24-hexaoxa-1,10-diazabicyclo[8.8.8]hexacos-5-ane(Ligand 33 from FIG. 3)

A solution of 0.608 g of 4-bromocatechole (1 equiv, 3.17 mmol) in 5 mLof acetone was added to a solution of methyl bromo ester (3 equiv, 0.9mL, 9.52 mmol) and potassium carbonate (3 equiv, 1.32 g, 9.52 mmol) in25 mL of acetone over a period of 5 min under an Ar atmosphere atreflux, and the resulting mixture was heated under reflux for 26 h underan Ar atmosphere. After the reaction, excess K₂CO₃ was filtered off anddried under reduced pressure. Purification was done using silica gelchromatography (1:1 hexanes/ethylacetate) to yield 0.900 g (98%) of asolid. This material (0.91 g, 3.15 mmol) and 0.36 g of Dowex50WX8-200was added to 31 mL of water and heated under reflux for 24 h under an Aratmosphere without stirring. The resulting mixture was filtered, and theresin was washed with MeOH. The filtrate was dried under reducedpressure to yield a white powdery solid 0.8 g (97%). Some of thismaterial (0.29 g, 0.95 mmol) was dissolved in thionyl chloride (5.0 mL,68 mmol) under Ar and was heated at reflux for 4 h. Excess thionylchloride was removed under reduced pressure, and the residue wasdissolved in anhydrous toluene (20 mL). The resulting solution and asolution of 1,4,10,13-tetraoxa-7,16-diazacyclooctadecane (0.251 g, 0.95mmol, 1.0 equiv) and triethylammine (0.38 mL, 2.85 mmol, 3.0 equiv) inanhydrous toluene (20 mL) were added simultaneously (50 mL/h) to aseparate flask containing anhydrous toluene (60 mL) at 0-5° C. under anAr atmosphere. The solution was stirred for 12 h at ambient temperature.An orange suspension formed and was filtered, and the solvent wasremoved under reduced pressure to yield a brown solid. Purification wasdone using silica gel chromatography (8:1 CH₂Cl₂/methanol) to yield0.367 g (73%) of a colorless oil. The resulting compound (0.367 g, 0.691mmol, 1 equiv) was dissolved in 15 mL anhydrous THF and to it was added20.5 mL of 1 M BH₃-THF (20.73 mmol, 30 equiv) slowly while stirringunder an Ar atmosphere at 0-5° C. When the addition was done, thesolution was warmed to room temperature and heated under reflux for 24h. Then the solution was treated first with 6 M HCl (15 mL) and secondwith water (15 mL) and heated under reflux for another 6 h. The solutionwas then basified with NH₄OH and the solvent was removed under reducedpressure. The salt was filtered out and dried again. Purification wasdone using silica gel chromatography (8:1 CH₂Cl₂/methanol) to yielded0.220 g (54%) of a white solid.

5,6-(4-methylbenzo)-4,7,13,16,21,24-hexaoxa-1,10-diazabicyclo[8.8.8]hexacos-5-ane(Ligand 35 from FIG. 3)

A solution of 0.990 g of 4-methylcatechole (1 equiv, 7.73 mmol) in 18 mLof acetone was added to a solution of methyl bromo ester (3 equiv, 2.2mL, 23.2 mmol) and potassium carbonate (3 equiv, 3.21 g, 23.2 mmol) in90 mL of acetone for a period of 15 min under an Ar atmosphere andreflux, and the resulting mixture was heated under reflux for 26 h underan Ar atmosphere. After the reaction, excess K₂CO₃ was filtered off anddried under reduced pressure. Purification was done using silica gelchromatography (1:1 hexanes/ethyl acetate) to yield 1.617 g (76%) of ayellow oil. This material (6.03 mmol) and 0.870 g of Dowex50WX8-200 wasadded to 60 mL of water and heated under reflux for 29 h under an Aratmosphere without stirring. The resulting mixture was filtered, and theresin was washed with MeOH. The filtrate was dried under reducedpressure to yield an orange powdery solid 1.669 g (97%). Some of thismaterial (0.449 g, 1.62 mmol) was dissolved in thionyl chloride (5 mL,68 mmol) under Ar and was heated at reflux for 4 h. Excess thionylchloride was removed under reduced pressure, and the residue wasdissolved in anhydrous toluene (20 mL). The resulting solution and asolution of 1,4,10,13-tetraoxa-7,16-diazacyclooctadecane (0.425 g, 1.62mmol, 1.0 equiv) and triethylammine (0.66 mL, 4.9 mmol, 3.0 equiv) inanhydrous toluene (20 mL) were added simultaneously (50 mL/h) to aseparate flask containing anhydrous toluene (60 mL) at 0-5° C. under anAr atmosphere. The solution was stirred for 18 h at ambient temperature.An orange suspension formed and was filtered, and the solvent wasremoved under reduced pressure to yield a solid. Purification was doneusing silica gel chromatography (8:1 CH₂Cl₂/methanol) to yield 0.480 g(64%) of a pale yellow solid. The resulted compound (0.480 g, 1.03 mmol,1 equiv) was dissolved in 20 mL anhydrous THF and was added 20 mL of 1 MBH₃-THF (20.60 mmol, 20 equiv) slowly while stirring under Ar atmosphereat 0-5° C. When the addition was done, the solution was warmed to roomtemperature and heated under reflux for 26 h. Then the solution wastreated first with 6M HCl (20 mL) and second with water (20 mL) andheated under reflux at 90° C. for another 14 h. The solution was thenbasified with NH₄OH and removed the solvent under reduced pressure. Thesalt was filtered out and dried again. Purification was done usingsilica gel chromatography (8:1 CH₂Cl₂/methanol) to yield 0.143 g (32%)of solid.

Carboxylated Europium Complex:

4-Isothiocyanato-1,2-benzenediacarboxylic acid (1 equiv) is dissolved incarbonate buffer (pH 9.8, 3 mL, 0.5 M). The resulting solution is addedto a solution containing the amine-substituted cryptand (LIGAND 36 fromFIG. 3, 1 equiv) in the same carbonate buffer (2 mL). Throughout thecombination of the two solutions, the pH is monitored via pH paper andkept between 9 and 10. The reaction solution is allowed to stir atambient temperature for 11 h. Dialysis is performed using a 100-500 Damembrane, including changing of the dialysis water 3 times over thecourse of 24 h. The mixture is transferred to a flask and solvent isremoved under reduced pressure. The resulting ligand has the followingformula:

This complex is mixed with one equivalent of EuCl₂ to produce the finalcomplex.

While exemplary embodiments are described above, it is not intended thatthese embodiments describe all possible forms of the invention. Rather,the words used in the specification are words of description rather thanlimitation, and it is understood that various changes may be madewithout departing from the spirit and scope of the invention.Additionally, the features of various implementing embodiments may becombined to form further embodiments of the invention.

1.-34. (canceled)
 35. A compound having formula II:

wherein: n, o are each independently 1 or 2; L₁ is a multi-dentateligand moiety that bonds to Eu; L₂ is a multi-dentate ligand moiety thatbonds to Gd; Xp are counter-ions to maintain charge neutrality; and Spis a spacer moiety that provides separation between Eu and Gd.
 36. Thecompound of claim 35 wherein L₁ is has formula III:

Y¹, Y², Y³, Y⁴, Y⁵, and Y⁶ are each independently O or S; R₁, R₂, R₃ areeach independently H, C₁₋₁₂ alkyl, C₁₋₁₂ fluoroalkyl, Cl, F, Br, I,N(R)₂, NH₂, CH₂CO₂H, nitro, cyano, or C₆₋₁₄ aryl, C₅₋₁₄ hetereoaryl, or5 and 6 membered rings formed by combining R₁ on adjacent carbon atomsor R₂ and R₃ on adjacent carbon atoms, ═O by combining R₁, R₂, or R₃ onthe same carbon atom, ═S by combining R₁, R₂, or R₃ on the same carbonatom, or ═NR by combining R₁, R₂, or R₃ on the same carbon atom; and Ris H or C₁₋₁₂ alkyl with the proviso that at least one R₁, R₂, or R₃ isa bond to Sp or has a bond to Sp.
 37. The compound of claim 35 whereinL₂ include a plurality of groups having the following formula:


38. The compound of claim 35 wherein L₂ includes a moiety having thefollowing formula:


39. The compound of claim 35 having formula 5:


40. The compound of claim 35 having the following formula: